CN-122003497-A - Novel Endo S2 mutant enzymes

Abstract

Novel Endo S2 mutant enzymes are provided which exhibit significantly enhanced transglycosylation activity and retain a substantial portion of the hydrolytic activity and are useful for glycochain reconstruction of polypeptides or proteins having N-glycans, such as antibodies, for example.

Inventors

• ZHAO YUE
• TAO WEIKANG
• WEN YUNPING
• FANG YAN
• LI YARAN
• LIU ZIQUN
• WANG HONG
• QIN KEN
• YANG YANG
• YING HUA

Assignees

• 上海齐鲁制药研究中心有限公司

Dates

Publication Date

20260508

Application Date

20241108

Priority Date

20231110

Claims (17)

1. An Endo S2 mutant enzyme having the sequence: (a) SEQ ID NO. 1 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO. 1, or (B) The mutant enzyme has a mutation at the following 1 or 2 amino acid positions R176 and/or D179 on the basis of the consecutive amino acids from position 38 to 819 of SEQ ID NO. 1, and has glycoside hydrolysis and/or glycosyltransferase activity.
2. The Endo S2 mutant enzyme according to claim 1, wherein the mutation is at least one selected from the group consisting of R176Y, D A.
3. A polynucleotide encoding the Endo S2 mutant enzyme of claim 1 or 2.
4. A vector comprising the polynucleotide of claim 3.
5. A host cell transformed with the vector of claim 4.
6. Use of an Endo S2 mutant enzyme according to claim 1 or 2 for the glycoengineering of an N-glycan containing polypeptide or protein.
7. Use according to claim 6 for remodelling the glycoform of an antibody, or for glycochain reconstruction of a glycoprotein, or for the preparation of a detection drug or ADC drug comprising an antibody.
8. A method for glycostrand remodeling of an N-glycan-containing polypeptide or protein using a one-step method comprising the steps of: (a) Introducing an Endo S2 mutant enzyme according to claim 1 or 2; (b) Introducing a N-glycan-containing polypeptide or protein as a substrate; (c) Providing a sugar chain linker donor, and (D) Carrying out glycoside hydrolysis of the N-glycan-containing polypeptide or protein using the Endo S2 mutant enzyme and transferring the sugar chain linker donor to the glycoside hydrolysis product to provide a new sugar chain engineered polypeptide or protein; The one-step process does not contain a purification step for the glycoside hydrolysate.
9. A method for performing sugar chain remodeling of an N-glycan-containing polypeptide or protein by a two-step method by introducing two enzymes for glycoside hydrolysis and neoglycosyl transfer, respectively, wherein the Endo S2 mutant enzyme according to claim 1 or 2 is used at least in one step of glycoside hydrolysis or neoglycosyl transfer, the method comprising the steps of: (a) Introducing an Endo S2 mutant enzyme according to claim 1 or 2 or other suitable endoglycosidase; (b) Introducing a polypeptide or protein comprising an N-glycan as a substrate and performing glycoside hydrolysis under the action of the enzyme of step (a) to provide a glycoside hydrolysate as a acceptor for the next step of converting a new glycosyl; (c) Providing a sugar chain linker donor, and (D) Transferring the sugar chain linker donor to the acceptor by said Endo S2 mutant enzyme or other suitable glycosyltransferase to produce a sugar chain engineered novel polypeptide or protein; Optionally, a purification step of the glycoside hydrolysis product is further included between steps (b) and (c).
10. The method of claim 8 or 9, wherein the N-glycan-containing polypeptide or protein comprises a naturally occurring antibody, a recombinant antibody, an Fc fragment of an antibody, or a sialoglycopeptide.
11. The method of claim 8 or 9, wherein the sugar chain linker donor is a synthetic or natural N-glycan oxazoline.
12. The method of claim 11, wherein the natural N-glycan oxazoline comprises a complex glycan oxazoline, a high mannose glycan oxazoline, or a hybrid oxazoline.
13. The method of claim 11, wherein the synthetic glycan oxazoline is a disaccharide oxazoline, a trisaccharide oxazoline, a tetrasaccharide oxazoline, a pentasaccharide oxazoline, a hexasaccharide oxazoline, a heptasaccharide oxazoline, an octasaccharide oxazoline, a nonasaccharide oxazoline, a decasaccharide oxazoline, or an undecsaccharide oxazoline.
14. The method of claim 8 or 9, wherein the sugar chain linker donor further comprises an additional modification method or an additional tag.
15. The method of claim 14, wherein the additional modification method is selected from azide, hydroformylation, alkynylation, sulfhydrylation, hydroxylation, carboxylation, phosphorylation, sialylation, albumin whitening, farnesylation, or modification conjugated to a polymer.
16. The method of claim 14, wherein the additional tag is selected from the group consisting of a therapeutic drug, a toxin substance, a fluorescent probe, biotin, a lipid, an antigen fragment, PEG, RNA, DNA, a polypeptide, a protein, an enzyme substrate, an enzyme inhibitor, an enzyme modulator, or an antibody fragment.
17. A sugar chain remodelled antibody and ADC medicament thereof prepared by the method of claims 8-16.

Description

Novel Endo S2 mutant enzymes The present application claims priority from China patent office, application No. CN2023115016001, china patent application entitled "novel Endo S2 mutant enzyme", filed 10.2023, 10.31, and China patent office, application No. CN202411548065X, china patent application entitled "novel Endo S2 mutant enzyme", which are incorporated herein by reference in their entirety. Technical Field The present disclosure relates to the fields of glycochemistry and enzymology, in particular to the use of recombinant novel Endo S2 mutant enzymes for glycoengineering polypeptides or proteins, which engineered proteins, such as antibodies, can be used for glycosite-directed conjugation of antibody drug conjugates. Background The Antibody Drug Conjugate (ADC) is characterized in that a connector is adopted to connect cytotoxin to an Antibody, so that the specific targeting effect of the Antibody on tumor-related antigens and the high-efficiency loading capacity of the cytotoxin are utilized to realize the targeted transportation and enrichment of the cytotoxin and accurately kill tumor cells. Therefore, ADC has lower side effects, broader therapeutic effects and higher therapeutic index than traditional chemotherapeutic drugs, and has become one of the fastest growing drugs in recent years. Traditional ADC coupling methods use lysine (Lys) or cysteine (Cys) in the antibody in high abundance and good exposure to bind to cytotoxins. This random coupling approach can lead to heterogeneity of ADC, i.e. non-uniform number of cytotoxin coupling sites, couplings, negatively affecting parameters such as pharmacokinetics, tolerability, availability, etc. The later developed site-directed coupling techniques, such as specific amino acid modification techniques (THIOMAB techniques), unnatural amino acid insertion techniques, sugar site-directed coupling techniques, enzyme-catalyzed coupling techniques, etc., have their advantages and disadvantages. A typical IgG antibody consists of two light chains, two heavy chains, four chains constituting three distinct domains, including two variable Fab domains (for recognition and binding to antigen) and one constant Fc domain (binding to fcγr on the surface of the recipient cell, mediating the interaction of the antibody with the host cell). Almost all therapeutic antibodies have an N-glycan modification of asparagine (N297) at position 297 in the Fc domain, and this conserved glycosylation site can be used as a coupling site for cytotoxins, and sugar site-directed coupling techniques have been developed based thereon. The sugar site-directed coupling technology has the advantages that the N297 glycan site of the Fc domain is far away from the Fab domain, so that the risk of the coupled antibody that the antigen binding capacity is reduced is avoided, and meanwhile, the amino acid sequence of the antibody is not required to be designed and modified by the coupling method, so that the risk of immunogenicity is reduced. Endoglycosidase, also called endo beta-N-acetylglucosaminidase, is a glycoside hydrolase acting on beta-1, 4-glycosidic bond, has become the research focus of antibody glycoengineering in recent years, and has also been found to have glycosyl transfer activity and can be used in ADC glycosite-directed coupling technology. Endoglycosidase sources are wide-ranging and include Endo S from streptococcus pyogenes (Streptococcus pyogenes), endo F1, endo F2, endo F3 from flavobacterium meningitidis (Flavobacterium meningosepticum), endo S2 from serotype M49 streptococcus pyogenes (Streptococcus pyogenes), and the like. Among them, endo S2 has a broader substrate specificity, and can recognize and act on complex glycans (Endo S cannot), high mannose glycans (Endo S cannot), complex multi-antenna glycans (Endo F1 cannot), core nonfucosylated glycans (Endo F3 cannot), core fucosylated glycans, so Endo S2 has great potential in the field of sugar site-directed coupling preparation of ADC. However, endo S2 belongs to the glycosidase GH18 family, more has glycoside hydrolysis activity, and has lower transglycosylation activity, and cannot be applied to the production of ADC. Thus, there is an urgent need to obtain Endo S2 mutants with significantly enhanced transglycosylation activity, retaining a substantial part of the hydrolytic activity, to facilitate antibody glycosylation remodelling and sugar site-directed conjugation using a one-step process to prepare ADC drugs. Summary of The Invention The present disclosure provides a recombinant Endo S2 mutant enzyme which exhibits enhanced transglycosylation activity while retaining a large part of hydrolysis activity, and is capable of glycoengineering a polypeptide or protein comprising N-glycans, including glycohydrolysis and glycostrand reconstruction, and thus is applicable to one-step glycosite-specific coupling of antibodies or Fc fragments thereof, for example, synthesis of ADC drugs or detection drugs comprising